Vol. 276, Issue 1, H141-H148, January 1999
Suppression of 
-adrenergic responsiveness of L-type
Ca2+ current by IL-1 in rat
ventricular myocytes
Shi J.
Liu1,2,
Weiguo
Zhou3, and
Richard H.
Kennedy2
Departments of
1 Biopharmaceutical Sciences,
2 Pharmacology and Toxicology, and
3 Anesthesiology, University of Arkansas for
Medical Sciences, Little Rock, Arkansas 72205
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ABSTRACT |
The possible mechanism by which interleukin-1
(IL-1) affects -adrenergic responsiveness of L-type
Ca2+ current
(ICa,L) was
examined in adult rat ventricular myocytes by use of whole cell
patch-clamp techniques. In the presence of isoproterenol (Iso),
exposure for 3 min to IL-1 suppressed the Iso-activated
ICa,L. In the
presence of IL-1, the response of ICa,L to Iso was
decreased, and the EC50 for Iso
stimulation was increased. However, IL-1 had no effect on
[3H]CGP-12177 binding,
displacement of
[3H]CGP-12177 binding
by Iso, or on basal and Iso-enhanced cAMP content. When
ICa,L was
activated by extracellular application of forskolin or
8-(4-chlorophenylthio)-cAMP, a membrane-permeable cAMP analog, or by
intracellular dialysis with cAMP, IL-1 had little effect on
ICa,L. In
contrast, in the presence of cAMP, IL-1 still suppressed the
Iso-enhanced
ICa,L. These
results show that the IL-1-induced decrease in -adrenergic
responsiveness of
ICa,L does not
result from inhibition of -adrenoceptor binding, adenylyl cyclase
activity, or cAMP-mediated pathways, suggesting a cAMP-independent mechanism.
cytokines; calcium channel; signal transduction; cardiac myocytes
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INTRODUCTION |
INTERLEUKIN-1 (IL-1), a 17-kDa proinflammatory
cytokine, has been closely associated with immune- and injury-mediated
changes in cardiovascular function (8, 9, 25, 33). Marked increases in
plasma IL-1 concentration are observed during the cardiac dysfunction associated with myocardial infarction (13, 29), ischemia-reperfusion (11, 15), myocarditis (6), acute septic cardiomyopathy (27), and allograft rejection (16). Studies using PCR
techniques show that mRNAs for IL-1 and its receptor are expressed
in endomyocardium of patients with inflammatory myocarditis (18) and
dilated cardiomyopathy (32) and in hearts with acute viral myocarditis
(24). The enhanced expression of IL-1 mRNAs was shown to occur
primarily in ventricular myocytes (24). Moreover, these cardiac
disorders are associated with an increased sympathetic nervous system
activity (22) and altered adrenergic responsiveness of myocardial
function (1, 6, 12, 14). The interaction between the increased IL-1
and the enhanced sympathetic tone under these pathophysiological
conditions remains unclear.
The direct autocrine and/or paracrine effects of IL-1 on
ventricular cell function include decreases in basal L-type
Ca2+ channel current
(ICa,L) (26)
and contractility (10, 20, 36). In addition, in neonatal rat cardiac
myocytes, IL-1 decreases the -adrenergic responsiveness of
contractility by reducing isoproterenol (Iso)-enhanced cAMP levels
after a 72-h exposure (14). Studies in adult guinea pig ventricular
myocytes suggest that preincubation with IL-1 for 1-5 h
inhibits the -adrenergic control of
ICa,L via
activation of nitric oxide synthase (NOS) (31). These data demonstrate
a delayed effect of IL-1 on myocardial -adrenergic responsiveness. However, available data have not addressed the possibility that IL-1 has an acute effect on adult ventricular myocyte responsiveness to -adrenergic stimulation.
In the present study we have examined the acute effect of IL-1 on
-adrenergic receptor binding and on intracellular cAMP content and
ICa,L in adult
ventricular myocytes. We show that IL-1 decreases the -adrenergic
responsiveness of
ICa,L primarily via a cAMP-independent pathway.
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METHODS |
Myocyte isolation.
Single adult ventricular myocytes were isolated from the hearts of male
Sprague-Dawley rats (250-300 g) with use of protocols described
previously (26). Briefly, hearts were rapidly excised and perfused at
37°C via the aorta with an oxygenated control buffer solution
consisting of (in mM) 110 NaCl, 3.8 KCl, 1.2 KH2PO4, 1.2 MgSO4, 25 NaHCO3, 0.2 CaCl2, and 11 glucose (pH 7.4 in
95% O2-5%
CO2 at 37°C) and for another 5 min with Ca2+-free buffer
solution. Hearts were then perfused for 20 min with a buffer solution
containing 25 µM CaCl2 plus 0.5 mg/ml collagenase. The ventricles were removed, minced, rinsed with
control buffer solution, and shaken in a water bath at 37°C for two
to three periods of 10 min each. Isolated ventricular myocytes were
then plated into 60-mm culture dishes (Falcon) containing
antibiotic-free, bicarbonate-buffered culture medium 199 (60%; GIBCO,
Grand Island, NY) with 36% Earle's balanced salt solution composed of
(mM) 116 NaCl, 4.7 KCl, 0.9 NaH2PO4,
0.8 MgSO4, 26 NaHCO3, and 5.6 glucose and 4%
fetal bovine serum (GIBCO; pH 7.4 in 5%
CO2-95% air at 37°C).
Electrophysiological measurements.
Ventricular myocytes were placed on the heated stage of an inverted
microscope (Nikon Diaphot) and perfused with a normal Tyrode solution
consisting of (in mM) 145 NaCl, 5.4 KCl, 0.8 MgCl2, 1.0 CaCl2, 5.6 glucose, 5.8 HEPES, and
4.2 Tris base (pH 7.4 at 37°C). Cells were patch clamped in the
whole cell configuration by conventional techniques (17) with use of a
patch-clamp amplifier (Axopatch 200A, Axon Instruments, Foster City,
CA), as previously described (26). Briefly, patch electrodes were
filled with a pipette solution; tip resistance was 2-5 M
.
Recorded currents were filtered at 1-2 kHz through a four-pole
low-pass Bessel filter and sampled at 5 kHz with a PC/AT computer using
pCLAMP 6.03 software (Axon Instruments) through an Axon Digidata 2000A
acquisition system.
Measurement of
ICa,L has been
described in our previous studies (26). The pipette solution for
experiments measuring
ICa,L consisted
of (in mM) 100 CsOH, 70 aspartic acid, 11 CsCl, 15 tetraethylammonium chloride, 2 MgCl2, 5 MgATP, 10 EGTA, 0.1 CaCl2, 5 pyruvic acid, 5.6 glucose, 5 Tris2-
phosphocreatine, 0.4 Li4GTP, and
10 HEPES-Tris base (pH 7.2 at 37°C). Myocytes were voltage clamped
at 
70 mV when the normal Tyrode solution was switched to an
external solution consisting of (in mM) 140 N-methyl-D-glucamine
chloride, 2 CaCl2, 0.8 MgCl2, 2 4-aminopyridine, and 10 HEPES-Tris base (pH 7.40 at 37°C). These conditions eliminated most
membrane currents associated with
Na+ and
K+. After formation of the whole
cell configuration,
ICa,L was
elicited by a single 250-ms pulse to +10 mV from the holding potential once every 15 s. The peak current-voltage relationship of
ICa,L was
constructed by applying 250-ms voltage pulses to potentials between
60 and +70 mV in 10-mV increments from the holding potential of
70 mV at 0.1 Hz. The magnitude of
ICa,L was defined
by the difference between the peak current and that at the end of the 250-ms pulse. All experiments were carried out at 37°C.
Membrane preparation.
-Adrenoceptor binding was performed using partially purified
membranes that were prepared at 4°C. Ventricular muscle from 8-10 rats was pooled, suspended in 50 mM Tris, 2 mM
MgSO4, 0.1 mM dithiothreitol, and
0.1 mM phenylmethylsulfonyl fluoride (pH 7.4), and initially
homogenized for 30 s using a Polytron at a setting of 6. Further
homogenization was achieved using seven strokes of a Dounce
homogenizer. The homogenate was centrifuged at 800 g for 20 min, and the supernatant was
subsequently centrifuged at 2,500 g
for 20 min. The resultant supernatant was subjected to two sequential
centrifugations at 30,000 g for 20 min
with use of the homogenizing buffer to wash the pellet between
centrifugations. The final pellet, a partially purified membrane
preparation, was resuspended in a reaction solution (50 mM Tris and 2 mM MgSO4, pH 7.4) and stored at
80°C. Protein concentrations were determined by the method
of Bradford (3), with BSA as the standard.
Radioligand binding to membrane preparations.
Binding assays with
[3H]CGP-12177 (42.5 Ci/mmol; New England Nuclear Research Products, Boston, MA) were
performed in polypropylene tubes containing reaction solutions in the
presence or absence of 4 ng/ml IL-1. In saturation experiments,
final concentrations of the -adrenergic antagonist
[3H]CGP-12177 ranged
from ~0.02 to 10 nM. In competition experiments, increasing levels of
nonlabeled Iso were added to reaction solutions containing 1.0 nM
[3H]CGP-12177 in the
presence or absence of 100 µM 5'-guanylyl imidodiphosphate guanosine (GppNHp). Nadolol (10 µM final concentration) was added to
a parallel set of tubes to estimate nonspecific binding in all
experiments. The reaction was initiated by addition of membrane protein
to assay tubes, and the contents were incubated at 37°C for 30 min.
Bound and free
[3H]CGP-12177 were
separated by filtration through GF/C filters, which were washed three
times with ice-cold reaction solution. Filters were then immersed in
scintillation fluid, and the retained radioactivity was determined by
liquid scintillation spectrometry. Data were analyzed using a
microcomputer version of LIGAND (28).
Radioligand binding to ventricular myocytes.
Effects of IL-1 on -adrenoceptor binding were also monitored in
intact ventricular myocytes (0.5 × 106 cells/ml normal Tyrode
solution containing 0.5 nM
[3H]CGP-12177) with
and without 4 ng/ml IL-1. Iso (100 nM) was included in some tubes,
and nonspecific binding was determined using
10
5 M nadolol. After 30 min
of incubation at 37°C, bound and free [3H]CGP-12177 were
separated by filtration through GF/C filters, which were washed three
times with ice-cold Tyrode solution. Radioactivity was determined as
described above. Data were analyzed using a microcomputer version of
LIGAND (28).
Chemicals and solutions.
Most reagents were purchased from Sigma Chemical (St. Louis, MO).
Nucleotides were directly added to pipette or bath solutions. The stock
solution of human recombinant IL-1
[106 U/ml (Promega, Madison,
WI) and 5 µg/ml (R & D Systems, Minneapolis, MN)] was made in
the normal Tyrode solution containing 0.1% BSA. The effect on
ICa,L of the
final concentration of 1,000 U/ml IL-1 (or 4 ng/ml) from Promega is
equivalent to that of 5 ng/ml IL-1 from R & D Systems.
Statistics.
Values are means ± SE. Statistical significance was evaluated by
the two-tailed Student's t-test or,
when more than two conditions are compared, by one-way ANOVA with
Duncan's multiple range test. Differences with
P < 0.05 were considered significant.
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RESULTS |
Suppression of -adrenergic responsiveness of
ICa,L by IL-1 in adult rat
ventricular myocytes.
Initial experiments were designed to examine the effect of IL-1 on
ICa,L during
stimulation by Iso. In Fig.
1A,
exposure of a myocyte to 50 nM Iso caused an 80% increase in
ICa,L that was
further enhanced (~60%) by subsequent exposure to 1 µM Iso. In the
presence of 1 µM ISO a 3-min exposure to 0.4 ng/ml IL-1 resulted
in an ~30% decrease in peak
ICa,L. The
ICa,L recovered after removal of IL-1 and return to 50 nM Iso. Similar experiments show that, in the presence of 1 µM Iso, 0.4 and 4 ng/ml, IL-1 decreased Iso-activated
ICa,L by 33.2 ± 4.7% (n = 6) and 43.1 ± 1.4% (n = 5; see also Fig.
6A), respectively. The 3-min
exposure duration was chosen because, during this period of time,
desensitization of -adrenergic responsiveness was minimal (~7%;
Fig. 1B). Figure 1B shows the temporal change in
ICa,L in the
presence and absence of IL-1. The result indicates that the
IL-1-induced decrease in Iso-activated
ICa,L was greater
than that observed in the continued presence of Iso.
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Fig. 1.
Effect of interleukin 1 (IL-1) on isoproterenol (Iso)-activated
L-type Ca2+ channel current
(ICa,L) in
adult rat ventricular myocytes. A:
exposure of a myocyte to 50 nM ( ) and 1 µM Iso ( ) resulted in
80 and 140% increases in
ICa,L,
respectively. Subsequent addition of 0.4 ng/ml IL-1 caused a 30%
reduction of
ICa,L in presence
of 1 µM Iso ( ).
ICa,L recovered
when IL-1 was removed and solution was returned to 50 nM Iso.  ,
Control Inset: superimposed current
traces where indicated; calibration bars, 10 pA/pF (vertical) and 5 ms
(horizontal); dashed line, 0-current level. Cell membrane capacitance
was 190 pF. B: time-dependent changes
in Iso-activated
ICa,L in absence
and presence of IL-1. After peak
ICa,L reached a
steady state in 1 µM Iso (designated time
0), current was monitored over time. Time-dependent
decay was obtained by plotting currents that were normalized to
magnitude of peak
ICa,L at
time 0 as a function of time and fit
by a straight line with linear regression. Slope of decline in
Iso-activated peak
ICa,L in absence
of IL-1 was 0.01 min1
(light dashed line, 21 experiments). Exposure to 0.4 ng/ml IL-1
caused a significant decrease in Iso-activated
ICa,L, with a
slope of 0.08 min1 (dark
dashed line, 13 experiments).
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Figure 2 demonstrates the effect of IL-1
on -adrenergic responsiveness of
ICa,L in adult
rat ventricular myocytes. Figure 2A
shows results from a myocyte that was pretreated with 0.4 ng/ml IL-1
for 3 min before exposure to increasing concentrations of Iso (1 nM-1 µM). In the presence of 0.4 ng/ml IL-1, 1 µM Iso induced a 58% increase in
ICa,L, a level
less than that observed in the absence of IL-1. The maximal
stimulation of
ICa,L by 1 µM
Iso was 192 ± 8% (n = 35) and
150 ± 3% (n = 24) of
control in the absence and presence of 0.4 ng/ml IL-1, respectively. Figure 2B shows the
concentration-dependent effects of Iso on ICa,L by plotting
relative peak
ICa,L (as
measured at +10 mV) to the value at 1 µM Iso vs. Iso concentrations
in the absence and presence of 0.4 ng/ml IL-1. IL-1 caused a
rightward shift in the dose-response curve, with the
EC50 for Iso being increased from
50.5 to 156 nM. Thus IL-1 reduced the maximal stimulation (efficacy)
and the potency of Iso.
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Fig. 2.
Effects of IL-1 on -adrenergic responsiveness of
ICa,L.
A: -adrenergic responsiveness of
ICa,L was
examined by exposing a myocyte to increasing concentrations of Iso
after a 3-min preincubation with 0.4 ng/ml IL-1.
Inset: superimposed current traces;
calibration bars, 10 pA/pF (vertical) and 5 ms (horizontal); dashed
line, 0-current level. Cell membrane capacitance was 175 pF.
B: concentration-response curves
showing effects of Iso on
ICa,L were
obtained in control conditions and in presence of 0.4 ng/ml IL-1.
Data were curve fit by Hill equation:
I = Imax · [Iso]h/([Iso]h + ECh50),
where I is peak current normalized to
that at 1 µM Iso,
Imax is peak
current at maximum effective concentration, [Iso], is Iso
concentration, h is Hill coefficient, and
EC50 is concentration of Iso
producing a half-maximal effect. Values are means ± SE; number of
experiments is in parentheses. Values for
EC50 and h were 50.5 ± 2.7 nM
and 1.1 in control and 156 ± 29 nM and 1.3 in presence of 0.4 ng/ml
IL-1 (estimated value ± SE of 95% confidence of curve
fitting).
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Effect of IL-1 on cardiac
-adrenergic receptor binding.
In attempts to determine the mechanism of the IL-1-induced decrease
in -adrenergic responsiveness, we first examined whether IL-1
affects -adrenoceptor binding. Figure 3
shows representative Scatchard plots from saturation binding assays
with use of
[3H]CGP-12177, a
-adrenergic antagonist, in partially purified membranes prepared
from rat ventricular myocardium. Results from repeated assays were
analyzed by the nonlinear curve-fitting program LIGAND (28) and
consistently showed the best fit to be a single population of
high-affinity binding sites with a dissociation constant
(Kd) of 0.29 ± 0.03 nM and a maximal binding site density (Bmax) of 54.2 ± 12.6 fmol/mg protein (n = 3), values
similar to those reported by others (5). Figure 3 also shows that
IL-1 (4 ng/ml) had no effect on
Kd (0.36 ± 0.04 nM, n = 3) or
Bmax (56.6 ± 12.0 fmol/mg
protein, n = 3).
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Fig. 3.
Representative Scatchard plots of
[3H]CGP-12177 binding
to partially purified membrane preparations from rat cardiac
ventricular muscle in absence and presence of 4 ng/ml IL-1. Data
were analyzed by LIGAND (with a linear regression of
r2 > 0.92).
Binding capacities and apparent dissociation constants were 57.9 fmol/mg protein and 0.3 nM in control (light dashed line) and 60.9 fmol/mg protein and 0.33 nM in IL-1 (dark dashed line),
respectively.
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Figure 4 shows results from experiments
examining competitive displacement of
[3H]CGP-12177 binding
by increasing concentrations of Iso. Figure 4A shows that, in the absence of
guanine nucleotides, Iso competition for
[3H]CGP-12177 binding
was best characterized by a two-binding-site model with relative
affinities (Ki)
of 5.8 ± 1.8 × 108 M (representing 80.3 ± 3.0% of total specific
[3H]CGP-12177 binding)
and 1.4 ± 0.5 × 105 M (19.6 ± 2.8% of
total specific binding). Analysis by LIGAND always suggested that a
two-site model was a better fit than a single-site model; however, in
two of the six experiments, the F
value did not indicate significant differences between the two models.
The presence of 4 ng/ml IL-1 did not significantly alter the effect
of Iso on
[3H]CGP-12177 binding;
observed Ki
values were 6.9 ± 2.7 × 108 M (77.3 ± 4.3% of
total specific binding) and 1.0 ± 0.4 × 105 M (22.7 ± 4.3% of
total specific binding). Figure 4B
shows that, in the presence of 100 µM GppNHp, Iso antagonized
[3H]CGP-12177 binding
from a single population of binding sites with
Ki values of 0.71 ± 0.31 × 106 M in
control conditions (n = 3) and 0.82 ± 0.23 106 M in the
presence of 4 ng/ml IL-1 (n = 3).
LIGAND analysis demonstrated a single-site model to be the best fit in
all experiments. These findings were supported by data obtained from
binding studies with intact ventricular myocytes. Table
1 shows that 4 ng/ml IL-1 did not alter
specific [3H]CGP-12177
binding (0.5 nM final concentration) to adult rat ventricular myocytes
in the presence or absence of 0.1 µM Iso. In summary, results of the
binding studies indicated that IL-1 does not affect agonist binding
to -adrenoceptors.
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Fig. 4.
Effects of IL-1 on Iso displacement of
[3H]CGP-12177 binding
to rat cardiac ventricular membranes.
A: effects of Iso on
[3H]CGP-12177 binding
in absence (light dashed line) and presence (dark dashed line) of 4 ng/ml IL-1. B: experiments
performed in A with 100 µM
5'-guanylyl imidodiphosphate guanosine. Data were normalized to
specific [3H]CGP-12177
binding observed in absence of Iso and presented as means ± SE for
3 experiments in each group. Data were curve fit using LIGAND (see
METHODS).
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Table 1.
Effects of IL-1 on specific [3H]CGP-12177
binding to adult rat ventricular myocytes in the presence and absence
of Iso
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Effects of IL-1 on cAMP levels.
Studies in neonatal rat cardiac myocytes have shown that cytokines
decrease -adrenergic responsiveness by suppressing the Iso-induced
increase in cell cAMP concentration (14). We determined whether this is
the case in adult rat ventricular myocytes by examining cell cAMP
levels in response to Iso in the absence and presence of IL-1. The
basal intracellular cAMP concentration in adult rat ventricular
myocytes was 4.73 ± 0.33 pmol/105 cells
(n = 13) or 8.03 ± 0.56 pmol/mg
protein, a value comparable to that reported by other investigators
(37). In Fig. 5, a 5-min incubation in 0.1 µM Iso approximately doubled the intracellular cAMP concentration.
Incubation for 5 or 10 min with 5 ng/ml IL-1 had no effect on basal
or Iso-enhanced cAMP levels. These results suggest that the
IL-1-induced decrease in -adrenergic responsiveness did not
result from alterations in Iso-stimulated intracellular cAMP
accumulation.
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Fig. 5.
Effect of IL-1 on intracellular cAMP content in adult rat
ventricular myocytes in absence and presence of Iso.
Left: cAMP contents were measured in
myocytes in control conditions and in presence of 5 ng/ml IL-1, 0.1 µM Iso, or IL-1 + Iso for 5 min.
Right: cells were treated with Iso
alone for 5 min or IL-1 alone for 10 min or preincubated with 5 ng/ml IL-1 for 5 min and then treated with 0.1 µM Iso for another
5 min. Values are means ± SE; number of experiments is in
parentheses. * P < 0.05 compared with control.
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Effects of IL-1 on the cAMP-dependent activation of
ICa,L.
Because IL-1 had no effect on Iso-enhanced cAMP content, we then
determined whether IL-1 alters
ICa,L activated
by forskolin (Fsk). Figure
6A shows
results from a control experiment in which 4 ng/ml IL-1 attenuated
Iso-enhanced
ICa,L by 42%. In
Fig. 6B, 1 µM Fsk caused an ~77%
increase in
ICa,L, a level
similar to that induced by 1 µM Iso. However, in contrast to the
effect observed with Iso, a 3-min exposure to 4 ng/ml IL-1 did not
significantly affect peak
ICa,L in the
presence of Fsk. A subsequent exposure to IL-1 also had no effect on
ICa,L, further
supporting the lack of its effect on Fsk-enhanced
ICa,L (Fig.
6B). Average peak
ICa,L in the Fsk
and Fsk + IL-1 conditions were 167 ± 5%
(n = 5) and 168 ± 5%
(n = 5) of control, respectively.
These results showed that IL-1 has no effect on Fsk-activated
adenylyl cyclase activity or downstream effects of cAMP.
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Fig. 6.
Effect of IL-1 on forskolin (Fsk)-activated
ICa,L.
A: control experiment. Addition of 4 ng/ml IL-1 3 min after incubation of a myocyte with 1 µM Iso ()
caused a 40% decrease in
ICa,L ().
B: 1 µM Fsk caused a 77% increase
in ICa,L ().
Two subsequent exposures to 4 ng/ml IL-1 had no effect on
Fsk-activated
ICa,L ().
ICa,L recovered
fully after removal of Fsk ( ).
Insets: superimposed current traces;
calibration bars in A and B, 10 pA/pF (vertical) and 5 ms (horizontal);
dashed lines, 0-current level. Cell membrane capacitance was 176 pF
(A) and 194 pF
(B).
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To further determine whether IL-1 interferes with cAMP-mediated
activation of
ICa,L, we
examined the effect of IL-1 on
ICa,L in myocytes
internally dialyzed with cAMP or extracellularly perfused with
8-(4-chlorophenylthio)-cAMP (CPT-cAMP), a membrane-permeable analog of
cAMP. In Fig.
7A, 10 µM cAMP in the pipette solution almost doubled the peak
ICa,L, a level
similar to that induced by 1 µM Iso. Exposure of the myocyte to 4 ng/ml IL-1 had no significant effect on the cAMP-enhanced
ICa,L. Averaged
current magnitude in IL-1 was 0.96 ± 0.02 (n = 4) and 0.97 ± 0.03 (n = 4) of the control peak
ICa,L in the
presence of 1 and 10 µM cAMP, respectively. Similarly, in a
representative experiment in Fig. 7B,
extracellular perfusion of a myocyte with 0.3 mM CPT-cAMP caused a
240% increase in peak
ICa,L. Subsequent
exposure to 5 ng/ml IL-1 did not significantly alter the
cAMP-activated
ICa,L. Averaged
current magnitude in IL-1 was 0.94 ± 0.02 (n = 4) of the control peak
ICa,L in the presence of CPT-cAMP. These results suggested that the IL-1-induced suppression of Iso-enhanced
ICa,L is not
mediated by inhibition of the cAMP-dependent activation of
Ca2+ channels. We then examined
whether IL-1 suppresses Iso-enhanced ICa,L in the
presence of cAMP. Figure 8 shows an
increase in ICa,L of 150% in the presence of 0.1 mM CPT-cAMP that was further enhanced by addition of 1 µM Iso. Under these conditions, exposure to 5 ng/ml
IL-1 caused an ~43% inhibition of Iso-stimulated
ICa,L. Results
from five experiments show that IL-1 reduced
ICa,L by 21.9 ± 5.2% in the presence of CPT-cAMP and Iso. Similarly, in myocytes
internally dialyzed with 10 µM cAMP, 5 ng/ml IL-1 decreased Iso-stimulated
ICa,L by 24.6 ± 6.1% (n = 3). These data
further support the suggestion that the IL-1-induced suppression of
-adrenergic responsiveness of
ICa,L is mainly
mediated by a cAMP-independent mechanism rather than antagonism of the
cAMP-induced activation of Ca2+
channels.
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Fig. 7.
Effect of IL-1 on cAMP-activated
ICa,L.
A:
ICa,L magnitude
in a myocyte internally dialyzed with 10 µM cAMP in pipette solution
was approximately twice that observed under control conditions (cf. in B). IL-1 (4 ng/ml) had no
effect on cAMP-activated
ICa,L ().
B:
ICa,L in a
myocyte extracellularly perfused with 0.3 mM
8-(4-chlorophenylthio)-cAMP (CPT-cAMP) was increased by 240% ().
Subsequent addition of 5 ng/ml IL-1 did not significantly affect
cAMP-activated
ICa,L ().
Insets: superimposed current traces;
calibration bars in A and
B, 10 pA/pF (vertical) and 5 ms
(horizontal); dashed lines, 0-current level. Cell membrane capacitance
was 132 pF (A) and 130 pF
(B).
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Fig. 8.
Effect of IL-1 on Iso-activated
ICa,L in presence
of cAMP. Activation of
ICa,L of 2.5-fold
in presence of 0.1 mM CPT-cAMP was further enhanced by addition of 1 µM Iso. Subsequent exposure to 5 ng/ml IL-1 suppressed
Iso-activated
ICa,L by 43%.
ICa,L recovered
after solution was returned to 0.1 mM CPT-cAMP.
Inset: superimposed current traces;
calibration bars, 10 pA/pF (vertical) and 10 ms (horizontal); dashed
line, 0-current level. Cell membrane capacitance was 155 pF.
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DISCUSSION |
We previously showed that IL-1 reduces basal peak
ICa,L in rat
ventricular myocytes (26). The present study demonstrates that IL-1
decreases the -adrenergic responsiveness of
ICa,L by
suppressing the maximal effect of Iso and increasing its
EC50. IL-1 does not alter basal
or Iso-induced cAMP levels, -adrenoceptor binding, or Fsk-stimulated
ICa,L and has
little, if any, effect on cAMP-activated
ICa,L. These
results suggest that the IL-1-induced acute inhibition of Iso's
effects on ICa,L
is mediated primarily at a site other than the
-adrenoceptor-adenylyl cyclase-protein kinase A pathway.
Comparison with findings observed in other cardiac myocytes.
Studies with neonatal rat cardiac myocytes have shown that a 72-h
incubation with activated splenocyte-conditioned medium or IL-1
inhibits the -adrenergic responsiveness of contractility by
suppressing the Iso-enhanced cAMP level (14). A consecutive study
showed that -adrenoceptor binding was unaltered; however, Fsk-stimulated cAMP concentrations were enhanced, whereas
Iso-stimulated cAMP content was decreased by 7% after a 24-h treatment
(6). In contrast, the present study in adult rat ventricular myocytes shows no effect of IL-1 on basal or Iso-enhanced cAMP levels. The
discrepancy in these results could be due to numerous factors, such as
different developmental stages, duration of incubation with IL-1, or
different cell populations in primary culture of neonatal cardiac
myocytes. Developmental differences in the -adrenergic and Fsk
responsiveness of
ICa,L have been
shown in rat (23) and rabbit ventricular myocytes (30).
Studies in adult guinea pig ventricular myocytes have shown that
IL-1 does not alter the -adrenergic responsiveness of
ICa,L unless the
exposure duration of the cytokine is >1 h (31). This IL-1-induced
inhibition of -adrenergic responsiveness of
ICa,L was
attenuated by replacement of
L-arginine with
D-arginine and by incubation
with an inhibitor of NOS, suggesting the involvement of the NOS pathway
(31). These investigators did not provide information about whether
IL-1 affects basal or Iso-enhanced cAMP content; however, they did
show that IL-1 has no effect on Fsk-activated
ICa,L, as
indicated by our data (Fig. 6B). In addition, the present results obtained from adult rat ventricular myocytes show that the IL-1-induced inhibition of -adrenergic responsiveness of
ICa,L occurs
after only a couple minutes of cytokine exposure (Figs.
1A and
6A), when cGMP production is not significantly altered (unpublished data). These data suggest that the
NOS pathway is not involved in this action. The cause of the discrepancy between these two studies is unclear but could be attributed to differences in species and/or experimental
conditions. Species variations in the -adrenergic and Fsk
responsiveness of
ICa,L in
ventricular myocytes have been reported (23, 30).
IL-1 and cAMP.
Studies in vascular smooth cells showed that IL-1 stimulates cAMP
but not cGMP production within 1 h (2). The increased cAMP has been
suggested to mediate the stimulation of expression of inducible NOS and
production of nitrite that causes vasodilatation (2, 35). Similarly,
cAMP has been shown to upregulate IL-1-induced inducible NOS mRNA
expression and nitrite production in neonatal rat cardiac myocytes
(21). In contrast, a study in decidual cells showed that low
concentrations of IL-1 increase the production of cAMP during a 24-h
exposure, whereas low concentrations of the cytokine (1 ng/ml) inhibit
cAMP production (7). This study suggested that cAMP does not mediate
the IL-1-induced stimulation of prostaglandin production. In
addition, in astrocytoma cells, IL-1 induces IL-6 release without
altering cAMP formation (4). Our present study showed that IL-1 does
not affect the basal or Iso-induced cAMP production in adult rat
ventricular myocytes. Therefore, data suggest that the role of cAMP in
the signal transduction mechanisms for IL-1 varies among species and
cell types.
Possible mechanisms.
Our results show that IL-1 has no effect on Iso-stimulated cAMP
content, -adrenoceptor binding, or
ICa,L in the
presence of Fsk. The minor inhibitory effect (<5%) of IL-1 in the
presence of intracellular or extracellular cAMP suggests that a
cAMP-independent mechanism is involved in the IL-1-induced
inhibition of Iso-activated ICa,L. This is
supported by results showing that, in the presence of cAMP,
ICa,L is further
increased by additional exposure to 1 µM Iso, and the Iso-induced
increase in ICa,L
is decreased by addition of IL-1. It has been suggested that Iso can
stimulate ICa,L
via a cAMP-independent pathway that involves direct regulation via a G
protein in adult rat ventricular myocytes (23). However, because the
relative contribution of this direct G protein cAMP-independent effect
of Iso on ICa,L
has been questioned (19), the role of this proposed pathway for the
observed IL-1-induced suppression of -adrenergic responsiveness
of ICa,L requires
further investigation. We previously showed that IL-1 stimulates the
production of ceramide and that ceramide mediates the IL-1-induced
suppression of basal ICa,L in adult
rat ventricular myocytes (34). It is very likely that ceramide may be
involved in this cAMP-independent pathway that suppresses the
Iso-activated
ICa,L.
In summary, IL-1 suppresses -adrenergic responsiveness of
ICa,L via a
cAMP-independent pathway. This pathway may include IL-1-stimulated
ceramide production, which counterbalances, rather than disrupts, the
Iso-stimulated cAMP-dependent pathway. This action may play an
important role in the reduced myocardial function observed during
various cardiac disorders associated with cell injury and immune and
inflammatory responses. It is also possible that the IL-1-induced
decrease in -adrenergic responsiveness plays a cardioprotective role
by reducing energy demand during the compensatory phase in cardiac dysfunction.
| |
ACKNOWLEDGEMENTS |
We thank Meei-Yueh Liu for excellent technical assistance.
| |
FOOTNOTES |
This work was supported in part by grants from the American Heart
Association/Arkansas Affiliate, the American Health Assistance Foundation, and the Office of Naval Research.
Present address of W. Zhou: Dept. of Anesthesiology, Baylor College of
Medicine, Houston, TX 77030.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: S. J. Liu, Dept. of Biopharmaceutical
Sciences, University of Arkansas for Medical Sciences, 4301 West
Markham St., MS 522-3, Little Rock, AR 72205.
Received 24 March 1998; accepted in final form 4 September 1998.
| |
REFERENCES |
1.
Barber, M. J.,
T. M. Mueller,
B. G. Davies,
R. M. Gill,
and
D. P. Zipes.
Interruption of sympathetic and vagal-mediated afferent responses by transmural myocardial infarction.
Circulation
72:
623-631,
1985[Abstract/Free Full Text].
2.
Boese, M.,
R. Busse,
A. Mülsch,
and
V. Schini-Kerth.
Effect of cyclic GMP-dependent vasodilators on the expression of inducible nitric oxide synthase in vascular smooth muscle cells: role of cyclic AMP.
Br. J. Pharmacol.
119:
707-715,
1996[Medline].
3.
Bradford, M. M.
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal. Biochem
72:
248-254,
1976[Medline].
4.
Cadman, E. D.,
D. D. Naugles,
and
C. M. Lee.
cAMP is not involved in interleukin-1-induced interleukin-6 release from human astrocytoma cells.
Neurosci. Lett.
178:
251-254,
1994[Medline].
5.
Cerbai, E.,
L. Guerra,
K. Varani,
M. Barbieri,
P. A. Borea,
and
A. Mugelli.
-Adrenoceptor subtypes in young and old rat ventricular myocytes: a combined patch-clamp and binding study.
Br. J. Pharmacol.
116:
1835-1842,
1995[Medline].
6.
Chung, M. K.,
T. S. Gulick,
R. E. Rotondo,
G. F. Schreiner,
and
L. G. Lange.
Mechanism of cytokine inhibition of -adrenergic agonist stimulation of cyclic AMP in rat cardiac myocytes.
Circ. Res.
67:
753-763,
1990[Abstract/Free Full Text].
7.
Cole, O. F.,
H. Seki,
M. G. Elder,
and
M. H. Sullivan.
Interleukin-1 independently stimulates production of prostaglandin E2 and cyclic AMP from human decidual cells.
Biochim. Biophys. Acta
1269:
139-144,
1995[Medline].
8.
Dinarello, C. A.
Interleukin-1 and its biologically related cytokines.
Adv. Immunol.
44:
153-205,
1989[Medline].
9.
Dinarello, C. A.
The proinflammatory cytokines interleukin-1 and tumor necrosis factor and treatment of the septic shock syndrome.
J. Infect. Dis.
163:
1177-1184,
1994.
10.
Evans, H. G.,
M. J. Lewis,
and
A. M. Shah.
Interleukin-1 modulates myocardial contraction via dexamethasone sensitive production of nitric oxide.
Cardiovasc. Res.
27:
1486-1490,
1993[Abstract/Free Full Text].
11.
Finkel, M. S.,
R. A. Hoffman,
L. Shen,
C. V. Oddis,
R. L. Simmons,
and
B. G. Hattler.
Interleukin-6 (IL-6) as a mediator of stunned myocardium.
Am. J. Cardiol.
71:
1231-1232,
1993[Medline].
12.
Gaide, M. S.,
R. J. Myerburg,
P. L. Kozlovskis,
and
A. L. Bassett.
Elevated sympathetic response of epicardium proximal to healed myocardial infarction.
Am. J. Physiol.
245 (Heart Circ. Physiol. 14):
H646-H652,
1983.
13.
Guillén, I.,
M. Blanes,
M.-J. Gómez-Lechón,
and
J. V. Castell.
Cytokine signaling during myocardial infarction: sequential appearance of IL-1 and IL-6.
Am. J. Physiol.
269 (Regulatory Integrative Comp. Physiol. 38):
R229-R235,
1995[Abstract/Free Full Text].
14.
Gulick, T.,
M. K. Chung,
S. J. Pieper,
L. G. Lange,
and
G. F. Schreiner.
Interleukin and tumor necrosis factor inhibit cardiac myocyte -adrenergic responsiveness.
Proc. Natl. Acad. Sci. USA
86:
6753-6757,
1989[Abstract/Free Full Text].
15.
Gurevitch, J.,
I. Frolkis,
Y. Yuhas,
Y. Paz,
M. Matsa,
R. Mohr,
and
V. Yakirevich.
Tumor necrosis factor-
is released from the isolated heart undergoing ischemia and reperfusion.
J. Am. Coll. Cardiol.
28:
247-252,
1996[Abstract].
16.
Halloran, P. F.,
S. M. Cockfield,
and
J. Madrenas.
The mediators of inflammation (interleukin 1, interferon-
, and tumor necrosis factor) and their relevance to rejection.
Transplant. Proc.
21:
26-30,
1989[Medline].
17.
Hamill, O. P.,
E. Neher,
B. Sakmann,
and
F. J. Sigworth.
Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches.
Pflügers Arch.
391:
85-100,
1981[Medline].
18.
Han, R. O.,
P. E. Ray,
K. L. Baughman,
and
A. M. Feldman.
Detection of interleukin and interleukin-receptor mRNA in human heart by polymerase chain reaction.
Biochem. Biophys. Res. Commun.
181:
520-523,
1991[Medline].
19.
Hartzell, H. C.,
and
R. Fischmeister.
Direct regulation of cardiac Ca2+ channels by G proteins: neither proven nor necessary.
Trends Pharmacol. Sci.
13:
380-385,
1992[Medline].
20.
Hosenpud, J. D.,
S. M. Campbell,
and
D. J. Mendelson.
Interleukin-1 induced myocardial depression in an isolated beating heart preparation.
J. Heart Transplant.
8:
460-468,
1989[Medline].
21.
Ikeda, U.,
K. Yamamoto,
M. Ichida,
F. Ohkawa,
M. Murata,
O. Iimura,
E. Kusano,
Y. Asano,
and
K. Shimada.
Cyclic AMP augments cytokine-stimulated nitric oxide synthesis in rat cardiac myocytes.
J. Mol. Cell. Cardiol.
28:
789-795,
1996[Medline].
22.
Jurevicius, J.,
and
R. Fischmeister.
Longitudinal distribution of Na+ and Ca2+ channels and -adrenoceptors on the sarcolemmal membrane of frog cardiomyocytes.
J. Physiol. (Lond.)
503:
471-477,
1997[Medline].
23.
Katsube, Y.,
H. Yokoshiki,
L. Nguyen,
and
N. Sperelakis.
Differences in isoproterenol stimulation of Ca2+ current of rat ventricular myocytes in neonatal compared to adult.
Eur. J. Pharmacol.
317:
391-400,
1996[Medline].
24.
Kelley, K. W.,
K. Hutchison,
R. French,
R. M. Bluthe,
P. Parnet,
R. W. Johnson,
and
R. Dantzer.
Central interleukin-1 receptors as mediators of sickness.
Ann. NY Acad. Sci.
823:
234-246,
1997[Medline].
25.
Kumar, A.,
V. Thota,
L. Dee,
J. Olson,
E. Uretz,
and
J. E. Parrillo.
Tumor necrosis factor and interleukin 1 are responsible for in vitro myocardial cell depression induced by human septic shock serum.
J. Exp. Med.
183:
949-958,
1996[Abstract/Free Full Text].
26.
Liu, S.,
and
K. D. Schreur.
G protein-mediated suppression of L-type Ca2+ current by interleukin-1 in cultured rat ventricular myocytes.
Am. J. Physiol.
268 (Cell Physiol. 37):
C339-C349,
1995[Abstract/Free Full Text].
27.
Löw-Friedrich, I.,
D. Weisensee,
P. Mitrou,
and
W. Schoeppe.
Cytokines induce stress protein formation in cultured cardiac myocytes.
Basic Res. Cardiol.
87:
12-18,
1992[Medline].
28.
Munson, P. J.,
and
D. Rodbard.
Ligand: a versatile computerized approach for characterization of ligand-binding systems.
Anal. Biochem.
107:
220-239,
1980[Medline].
29.
Neumann, F.-J.,
I. Ott,
M. Gawaz,
G. Richardt,
H. Holzapfel,
M. Jochum,
and
A. Schömig.
Cardiac release of cytokines and inflammatory responses in acute myocardial infarction.
Circulation
92:
748-755,
1995[Abstract/Free Full Text].
30.
Osaka, T.,
and
R. W. Joyner.
Developmental changes in the -adrenergic modulation of calcium currents in rabbit ventricular cells.
Circ. Res.
70:
104-115,
1992[Abstract/Free Full Text].
31.
Rozanski, G. J.,
and
R. C. Witt.
IL-1 inhibits -adrenergic control of cardiac calcium current: role of L-arginine/nitric oxide pathway.
Am. J. Physiol.
267 (Heart Circ. Physiol. 36):
H1753-H1758,
1994[Abstract/Free Full Text].
32.
Satoh, M.,
G. Tamura,
I. Segawa,
A. Tashiro,
K. Hiramori,
and
R. Satodate.
Expression of cytokine genes and presence of enteroviral genomic RNA in endomyocardial biopsy tissues of myocarditis and dilated cardiomyopathy.
Virchows Arch.
427:
503-509,
1996[Medline].
33.
Schöbitz, B.,
E. R. De Kloet,
and
F. Holsboer.
Gene expression and function of interleukin 1, interleukin 6 and tumor necrosis factor in the brain.
Prog. Neurobiol.
44:
397-432,
1994[Medline].
34.
Schreur, K. D.,
and
S. Liu.
Involvement of ceramide in inhibitory effect of IL-1 on L-type Ca2+ current in adult rat ventricular myocytes.
Am. J. Physiol.
272 (Heart Circ. Physiol. 41):
H2591-H2598,
1997[Abstract/Free Full Text].
35.
Scott-Burden, T.,
E. Elizondo,
T. Ge,
C. M. Boulanger,
and
P. M. Vanhoutte.
Simultaneous activation of adenylyl cyclase and protein kinase C induces production of nitric oxide by vascular smooth muscle cells.
Mol. Pharmacol.
46:
274-282,
1994[Abstract].
36.
Weisensee, D.,
J. Bereiter-Hahn,
W. Schoeppe,
and
I. Löw-Friedrich.
Effects of cytokines on the contractility of cultured cardiac myocytes.
Int. J. Immunopharmacol.
15:
581-587,
1993[Medline].
37.
Zheng, J.-S.,
A. Christie,
M. B. De Young,
M. N. Levy,
and
A. Scarpa.
Synergism between cAMP and ATP in signal transduction in cardiac myocytes.
Am. J. Physiol.
262 (Cell Physiol. 31):
C128-C135,
1992[Abstract/Free Full Text].
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Abstract
Introduction
